Mechanism Of Intracellular Osmotic Pressure Modulating Intermediate Filaments Tension And The Growth Of Neurite

Objective:The polarization and growth of neurite are accompanied by many mechanical activities.NGF(Nerve growth factor)is a neurotrophic factor that has the dual biological functions of neuron nutrition and promotes the growth of neuronal processes.However,there are few reports on how NGF participates in the polarization and growth of neurons through intracellular mechanical activities.We applied the principle of fluorescence resonance energy transfer(FRET)to construct an angular structure tension detection probe,quantitative cytoskeletal tension,combined with the detection and quantification of intracellular protein colloids and ion osmotic pressure,In order to reveal the mechanical effects of cytoskeletal tension and intracellular osmotic pressure on neuron polarization and axonal growth during neural cell differentiation and related mechanisms.Then clarify the pharmacological mechanism of the traditional Chinese medicine monomer astragalus polysaccharide to improve the growth of neurons and axons by regulating the changes of intracellular tension.Methods:NGF stimulates neuron-like PC 12 cells to establish the models of neurite growth.And transfected four cytoskeleton fluorescent tension probes(AcpA,cpT,VcpV,GcpG)of actin,tubulin,vimentin and GFAP,Through FRET to convert the intracellular skeletal tension into optical signals,and measure the changes in the tension of various skeletal proteins in the cell under different stimuli;Total osmotic pressure of cells was measured by freezing osmotic pressure meter,Explore the effect of osmotic pressure on vimentin tension;Laser particle size analyzer records the size and number of cytoplasmic protein nanoparticles,and studies the changes of intracellular potential energy;Chloride Ion Fluorescence Probe(MQAE)and Calcium Ion Fluorescent Probe(Fluo-4AM)to detect the change of Cl-and Ca2+in pc 12 cells under different stimuli;Microtubule-associated protein(TAU)and Microtubule-associated protein2(MAP2)was used to mark the axons and dendrites of rat neurons in the cerebral cortex respectively,and observed by immunofluorescence.Results:1.In the NGF-induced nerve growth model,The microfilament force at the tip of the neurite was significantly weakened,while the tension of the intermediate fibers vimentin and GFAP increased significantly,while no microtubule tension was detected.2.NGF can up-regulate the tension of microfilaments and vimentin in axons,and induce the increase of intracellular osmotic pressure,The intracellular cytoplasmic protein nanoparticles are mainly distributed at 10nm,and the intracellular chloride and calcium ions increase,accompanied by primary neurons,Increased length of protrusions;3.Microfilament stabilizers can significantly reduce the tension and osmotic pressure of vimentin.Nanoparticles generated in the cytoplasm are mainly distributed at 100-1000nm,and the content of chloride and calcium ions is reduced,and the length of neuron protrusion is suppressed;4.Slingshot inhibitors(Sennosides A)reduce the tension and osmotic pressure of vimentin,reduce chloride ions,and decrease the length of neuronal processes;5.LIMK inhibitor BMS-5 attenuates vimentin tension and cytoplasmic osmotic pressure,inhibits the content of chloride and calcium ions in cells and the length of neuronal processes;6.Stimulate PC 12 cells with sodium chloride and sodium gluconate under the premise of Intracellular fluid mimic,Vimentin tension decreases with increasing concentrations of NaCl and Na+ gluconate,After mixing 50 mg/ml BSA,the downward trend of vimentin tension was significantly suppressed.At the same time,as the concentrations of extracellular NaCl and Na+ gluconate increased,the osmotic pressure of the solution also increased;7.Astragalus polysaccharide(APS)can up-regulate vimentin tension and Intracellular osmotic pressure and promote the increase of neurite length.Conclusion:This study explores the mechanical effects of cytoskeletal proteins on the growth of neuronal processes by NGF.Vimentin tension is enheanced at the tip of neurite of PC 12 cell,and regulated by the osmotic pressure of protein nanoparticles generated by NGF-induced microfilament depolymerization.Intracellular osmotic pressure and vimentin tension synergistically promote the growth of neuronal cell processes.At the same time,astragalus polysaccharides can participate in the growth of nerve cells by regulating vimentin tension and osmotic pressure.APS can participate in the growth of neuron by regulating vimentin tension and osmotic pressure.This study explores the mechanical activities of cells during the process of neuronal polarization and neurite outgrowth from the perspective of cytoskeletal tension,which provides deeper insights for the treatment of neurological diseases and functional recovery.